How to Combine STED and CLARITY

STED nanoscopy in optically cleared kidney tissue

Previously, the preferred way to study the subtlest elements of the kidney, such as foot processes and the slit diaphragm has been by the use of electron microscopy. Using STED microscopy, we show that the nanoscale localization of slit diaphragm proteins can now be resolved using light microscopy. Even if the nanoscopic resolution has been available for a decade, light microscopy studies of the slit diaphragm are not found in the literature. This is likely due to the difficulties of achieving the high quality of fluorescent labelling needed for super-resolution microscopy.

By applying an optical clearing protocol based on the CLARITY technique, we found that the immunostaining quality in kidney tissue can be improved. The improvement is likely due to the removal of lipids, resulting in a higher availability of binding epitopes in cleared tissue, as compared to PFA fixed non-cleared tissue. The optically cleared tissue, mounted in a refractive index matching saturated fructose solution, also gives the opportunity to perform 3D STED imaging at depths of at least 30 µm without the need for spherical aberration corrections. This allows for super-resolved studies of foot process morphology and protein topology on a 3D perspective in intact tissue, which could enhance our understanding regarding pathologies occurring in the kidney. In summary, we report that hydrogel-based optical clearing is not only beneficial for the study of larger samples on the millimeter-scale, but also on the nano-scale by improving the staining efficiency, a parameter that is crucial for super-resolved STED microscopy.